As a result of the RV144 trial and numerous recent studies, heterologous prime-boost HIV vaccine strategies have emerged as the leading approach to elicit immune responses that could protect against HIV infection. The molecular basis of the heterologous approach over homologous approaches is not well understood, however. In particular, immune responses to vector or DNA priming immunizations that are then amplified by protein boost immunization are complex and obscure. In this proposal, we will perform experiments that may clarify unsuspected, fundamental aspects of the immune responses to the prime that are amplified by the protein boost. To test this, we will control for many factors in a heterologous prime-boost experiment, varying only the conformational B-cell epitopes between prime and boost. Our innovative hypothesis is that the conformation of B-cell epitopes in the prime influences those self-same epitopes in the boost. Rabbits will be immunized with an array of DNA primes constructed to express or exclude specific immunogenic HIV variable loop epitopes and then boosted with an immunofocused protein immunogen that exclusively bears those epitopes. We expect to observe whether the expression of a conformational epitope in the prime amplifies or retards elicitation of neutralizin antibodies from that self-same epitope provided on the protein boost. The results will clearly provide the first consistent rationale for selecting or designing HIV env genes for use in priming immunizations. This exploratory R21 project will thus form the foundation for a unique long-term research program into the molecular details of the interaction between priming and boosting immunizations in a heterologous prime-boost strategy. These details may be crucial for designing an efficacious HIV vaccine.